Protection against CNS ischemia by temporary interruption of function-related processes of neurons.

Previous studies have shown that most of the energy consumption of CNS tissue is used for processes that subserve signaling functions of the cells. Since these function-related processes are probably not essential to cell viability, blocking them reversibly with a combination of pharmacologic agents should protect cells from a reduction in energy metabolism. Preliminary experiments to test this hypothesis were performed on isolated rabbit retinas. They were maintained in a newly devised chamber that permitted continuous monitoring of electrophysiological function for > or = 8 h. Ischemia was simulated by a 6-fold reduction in both O2 and glucose. This caused a rapid (t1/2 75 s) and complete loss of the light-evoked response in the optic nerve. Untreated retinas showed full recovery after 1/2 h of deprivation, but only 50% recovery after 1 h and little or no recovery after 2 or 3 h. Retinas exposed during 3 h of deprivation to a combination of six agents that abolished electrophysiologic function and reduced glucose utilization [tetrodotoxin (TTX), 2-amino-4-phosphonobutyric acid (APB), 2-amino-5-phosphonovaleric acid (APV), amiloride, Mg2+, and Li+] showed full recovery. We conclude that reducing energy requirements by blocking functional processes can prevent ischemic damage.

Importance of nitric oxide synthase inhibition to the attenuated vascular responses induced by topical L-nitroarginine during vibrissal stimulation.

We assessed the regional cerebral blood flow (rCBF) response to vibrissal stimulation before and after nitric oxide synthase (NOS) inhibitors were topically applied through a closed cranial window placed over the cortical barrel fields in anesthetized Sprague-Dawley rats. In the presence of L-nitroarginine (1 mM), both the maximum and total responses became reduced, but only in those animals demonstrating > 50% inhibition of NOS activity as determined by the conversion of [3H]arginine to [3H]citrulline within homogenates taken from cortical gray matter under the cranial window. The degree of enzyme inhibition depended in part, upon duration after topical application of NOS inhibitor. When > 50%, enzyme inhibition correlated with the decrease in maximum and total rCBF response (p < 0.01). These findings emphasize the merits of assessing enzyme activity after administering NOS inhibitors, and suggest that NO generated from parenchymal NOS activity plays an important role in the cerebrovascular response to physiologic somatosensory stimulation under the stated conditions.

Cerebral blood flow changes during cortical spreading depression are not altered by inhibition of nitric oxide synthesis.

CBF increases concomitantly with cortical spreading depression (CSD). We tested the hypothesis that CBF changes during CSD are mediated by nitric oxide (NO). Male Wistar rats (n = 23) were subjected to KCl-induced CSD before and after administration of nitric oxide synthase (NOS) inhibitors N-nitro-L-arginine (L-NNA) or N-nitro-L-arginine methyl ester (L-NAME) and in nontreated animals. CBF, CSD, and mean arterial blood pressure were recorded. Brain NOS activity was measured in vitro in control, L-NNA, and L-NAME-treated rats by the conversion of [3H]arginine to [3H]citrulline. Our data show that the NOS inhibitors did not significantly change regional CBF (rCBF) during CSD, even though cortical NOS activity was profoundly depressed and systemic arterial blood pressure was significantly increased. Our data suggest that rCBF during CSD in rats is not regulated by NO.

Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death.

Neuronal injury may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programmed cell death (PCD). Although the nature of this injury may be both preventable and reversible, the underlying mechanisms that mediate PCD are not well understood. Using the agent nicotinamide as an investigative tool in primary rat hippocampal neurons, the authors examined the ability to modulate two independent components of PCD, namely the degradation of genomic DNA and the early exposure of membrane phosphatidylserine (PS) residues. Neuronal injury was determined through trypan blue dye exclusion, DNA fragmentation, externalization of membrane PS residues, cysteine protease activation, and the measurement of intracellular pH (pHi). Exposure to the NO donors SIN-1 and NOC-9 (300 micromol/L) alone rapidly increased genomic DNA fragmentation from 20 +/- 4% to 71 +/- 5% and membrane PS exposure from 14 +/- 3% to 76 +/- 9% over a 24-hour period. Administration of a neuroprotective concentration of nicotinamide (12.5 mmol/L) consistently maintained DNA integrity and prevented the progression of membrane PS exposure. Posttreatment paradigms with nicotinamide at 2, 4, and 6 hours after NO exposure further demonstrated the ability of this agent to prevent and reverse neuronal PCD. Although not dependent upon pHi, neuroprotection by nicotinamide was linked to the modulation of two independent components of neuronal PCD through the regulation of caspase 1 and caspase 3-like activities and the DNA repair enzyme poly(ADP-ribose) polymerase. The current work lays the foundation for the development of therapeutic strategies that may not only prevent the course of PCD, but may also offer the ability for the repair of neurons that have been identified through the loss of membrane asymmetry for subsequent destruction.

Possible origins and distribution of immunoreactive nitric oxide synthase-containing nerve fibers in cerebral arteries.

The distribution of perivascular nerve fibers expressing nitric oxide synthase (NOS)-immunoreactivity was examined in Sprague-Dawley and Long-Evans rats using affinity-purified rabbit antisera raised against NOS from rat cerebellum. NOS immunoreactivity was expressed within the endothelium and adventitial nerve fibers in both rat strains. Labeled axons were abundant and dense in the proximal anterior and middle cerebral arteries, but were less numerous in the caudal circle of Willis and in small pial arteries. The sphenopalatine ganglia were the major source of positive fibers in these vessels. Sectioning postganglionic parasympathetic fibers from both sphenopalatine ganglia reduced the density of NOS-immunoreactive (IR) nerve fibers by > 75% in the rostral circle of Willis. Moreover, NOS-IR was present in 70-80% of sphenopalatine ganglion cells. Twenty percent of these neurons also contained vasoactive intestinal polypeptide (VIP)-immunoreactivity. By contrast, the superior cervical ganglia did not contain NOS-IR cells. In the trigeminal ganglion, NO-IR neurons were found chiefly within the ophthalmic division; approximately 10-15% of neurons were positively labeled. Colocalization with calcitonin gene-related peptide (CGRP) was not observed. Sectioning the major trigeminal branch innervating the circle of Willis decreased positive fibers by < or = 25% in the ipsilateral vessels. In the nodose ganglion, 20-30% of neurons contained NOS-immunoreactivity, whereas less than 1% were in the C2 and C3 dorsal root ganglia. Three human circles of Willis obtained at autopsy showed sparse immunoreactive fibers, chiefly within vessels of the posterior circulation. Postmortem delay accounted for some of the reduced density. Our findings indicate that nerve fibers innervating cerebral arteries may serve as a nonendothelial source of the vasodilator nitric oxide (NO). The coexistence of NOS and VIP within sphenopalatine ganglion cells raises the possibility that two vasodilatory agents, one, a highly diffusable short-lived, low-molecular-weight molecule, and the other, a polar 28 amino acid-containing peptide, may serve as coneuromediators within the cerebral circulation.

Calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity in endothelial cells after long-term stimulation of perivascular nerves.

Since both perivascular nerves and the endothelium are involved in the control of the tone of smooth muscle in the wall of blood vessels, we decided to test whether electrical stimulation of perivascular nerves might lead to structural and immunocytochemical changes in the vascular endothelium. Long-term electrical stimulation in vivo of perivascular nerves of the rabbit central ear artery was applied. The results indicate that chronic stimulation of perivascular nerves for 10 days induced structural changes and the appearance of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity in a subpopulation of endothelial cells.

Evoked noradrenaline release in the rabbit ear artery: enhancement by purines, attenuation by neuropeptide Y and lack of effect of calcitonin gene-related peptide.

1. Adenosine (30 microM) and its analogues 5'-N-ethylcarboxaminoadenosine (5 and 30 microM) and L-phenylisopropyladenosine (5 and 30 microM), potentiated the evoked but not spontaneous release of tritiated noradrenaline in the rabbit central ear artery. 2. Prejunctional inhibition of the evoked but not spontaneous release of tritiated noradrenaline by 100 nM neuropeptide Y is greater at 2 min than at 10 min after superfusion of the peptide. 3. Calcitonin gene-related peptide (2.63 to 263 nM) did not affect the evoked or spontaneous release of tritiated noradrenaline in this preparation. 4. These results are discussed in terms of prejunctional modulation of sympathetic transmission in the rabbit central ear artery.

Changes in purinergic responses of the rabbit isolated central ear artery after chronic electrical stimulation in vivo.

1. The effect of chronic (4-16 days) electrical stimulation (5 Hz, 0.3 ms, 4-10 V) of the great auricular nerve in vivo on sympathetic cotransmission in the rabbit isolated central ear artery was examined. 2. Chronic stimulation had no significant effect on frequency-dependent (4-60 Hz) neurogenic contractions or contractile responses induced by exogenous noradrenaline (0.1-300 microM). 3. In contrast, contractions induced by exogenous alpha, beta-methylene ATP (10.0 microM) were significantly decreased in preparations from 16-day stimulated animals in comparison with sham-operated, 4-day and 8-day chronically stimulated animal groups. 4. It is concluded that chronic electrical stimulation of nerves supplying the ear artery may lead to the selective alteration of postjunctional P2x-purinoceptor mechanisms, while the effects mediated by post-junctional alpha 1-adrenoceptors remain unchanged.

Sympathetic neurotransmission in the rabbit isolated central ear artery is affected as early as one week following a single dose of X-irradiation.

1. The short-term effect of a single dose of 4500 rad X-irradiation on sympathetic neurotransmission (involving both noradrenergic and purinergic components) was assessed in the rabbit central ear artery, 1, 4 and 6 weeks post-irradiation. 2. Neurally mediated contractions were reduced as early as 1 week post-irradiation, with responses to lower frequency stimulation being initially most affected. This suggest that the purinergic component of the contractile response is affected earlier than the adrenergic component. 3. There was no change in the amplitude or sensitivity of treated preparations to the cumulative application of noradrenaline when compared with untreated preparations. In contrast, contractions to alpha, beta-methylene ATP (1 microM), a P2-purinoceptor agonist, were significantly increased at 4 and 6 weeks post-irradiation, although not at 1 week post-irradiation. 4. There were no apparent changes in the pattern of catecholamine fluorescence as a result of irradiation. However, the tissue content of noradrenaline was significantly reduced 6 weeks post-irradiation when compared with control preparations. 5. It is concluded that damage to sympathetic cotransmission is one of the early effects of irradiation, with initial impairment predominantly of the purinergic component.

X-irradiation attenuates relaxant responses in the rabbit ear artery.

1. The relaxant actions of acetylcholine, substance P and calcitonin gene-related peptide, and the levels of neuropeptide Y and calcitonin gene-related peptide were assessed in the rabbit central ear artery 1, 4 and 6 weeks after a single dose of 45 Gy X-irradiation, a dose similar to that used clinically in intraoperative radiotherapy. 2. Relaxant responses induced by acetylcholine and substance P (both endothelium-dependent) and calcitonin gene-related peptide (endothelium-independent) were reduced, and endogenous neuropeptide Y and calcitonin gene-related peptide levels were unaffected after X-irradiation. 3. The mechanism(s) by which a single dose of 45 Gy X-irradiation may selectively damage relaxant, but not direct, contractile responses of the smooth muscle (as we have shown previously) of the rabbit central ear artery are discussed.

Gacyclidine (Beaufour-Ipsen).

Beaufour-Ipsen is developing gacyclidine (GK-11) for the potential treatment of traumatic brain injury. Phase II clinical trials of the compound for this indication had been completed as of October 1999 and the company is looking for a partnership before undertaking further clinical development for this and, possibly, other indications [344879], [346265], [386763]. Phase II trials for acute spinal cord injury gave disappointing results and development for this indication has been discontinued [344879].

Delayed multidose treatment with nicotinamide extends the degree and duration of neuroprotection by reducing infarction and improving behavioral scores up to two weeks following transient focal cerebral ischemia in Wistar rats.

A single, delayed dose of nicotinamide (NAm) was shown to be protective against focal cerebral ischemia in rats, but the protection was limited to three to seven days following stroke. The investigation reported here was conducted to examine if the use of multiple doses of NAm, administered after the onset of focal cerebral ischemia, would extend the duration of neuroprotection compared with a single dose treatment regimen. Male Wistar rats were subjected to transient focal cerebral ischemia by occluding the right middle cerebral artery (MCAo) for two hours. Following MCAo, motor and sensory behavioral tests were performed daily and the cerebral infarct volumes were measured at two weeks after sacrifice. Each animal was placed into one of four groups that received either normal saline alone (Group S), one (Group A), two (Group B), or three (Group C) doses of NAm (500 mg/kg). Each animal, therefore, received three treatments over two weeks, with the first dose administered intravenously two hours after the onset of MCAo. Single and multiple doses of NAm reduced the infarction (p < 0.01) and improved (p < 0.05) the neurologic sensory and motor behavior when compared with the saline-treated animals up to two weeks after stroke. Moreover, animals that received multiple doses of NAm recuperated full motor function not different from normal, preoperative motor behavior. Delayed treatment with NAm given as multiple doses, therefore, further enhances the extent and duration of neuroprotection by significantly reducing cerebral infarct volumes, improving neurologic behavioral scores, and confers a complete motor recovery up to two weeks from the onset of focal cerebral ischemia in Wistar rats.

Neuroprotection against ischemia by metabolic inhibition revisited. A comparison of hypothermia, a pharmacologic cocktail and magnesium plus mexiletine.

Previous studies have suggested that metabolic inhibition is neuroprotective, but little evidence has been provided to support this proposal. Using the in vitro rabbit retina preparation as an established model of the central nervous system (CNS), we measured the rate of glucose utilization and lactate production, and the light-evoked compound action potentials (CAPs) as indices of neuronal energy metabolism and electrophysiologic function, respectively. We examined the effect of three (3) treatments options: hypothermia (i.e., 33 degrees C and 30 degrees C), a six-member pharmacologic "cocktail" (tetrodotoxin (0.1 microM), 2-amino-4-phosphonobutyric acid (20 microM), 2-amino-5-phosphonovaleric acid (1 mM), amiloride (1 mM), magnesium (10 mM) and lithium (10 mM) and the combination of magnesium (Mg2+ 1 mM) and mexiletine (Mex, 300 microM) on in vitro rabbit retinas, to see if there is a correlation between neuronal energy metabolism during ischemia (simulated by the reduction of oxygen from 95% to 15% and glucose from 6 mM to 1 mM), and the subsequent recovery of function. Hypothermia and the "cocktail" significantly inhibited both the rate of glucose utilization and lactate production, whereas Mg2+ and/or Mex showed only a nonsignificant tendency toward a reduction, compared to control retinas. Recovery of light-evoked CAPs was significantly improved in hypothermia- and cocktail-treated retinas, as well as with retinas exposed to the combination of Mg2+ plus Mex, but not with Mg2+ or Mex alone, relative to control retinas. A linear regression analysis of the % recovery of function versus the % reduction in the rate of glucose utilization during ischemia showed a significant correlation (r2 = 0.80, correlation coefficient = 0.9, p < 0.05) between these two parameters. This and other data discussed provide convincing evidence that there is a correlation between metabolic inhibition, achieved during ischemia, and neuroprotection.

Nitric oxide modulates light-evoked compound action potentials in the intact rabbit retina.

This study examines the effects of alterations in nitric oxide (NO) production on rabbit retinal function. NG-nitro-L-arginine (L-NA) and L-arginine (L-Arg) inhibited the light-evoked compound action potentials (CAPs) from the optic nerve, but not the electroretinogram (ERG), in a concentration-dependent manner, whereas D-arginine had no effect. L-Arg partially reversed the L-NA-induced inhibitory effect, and L-NA prevented the L-Arg-induced attenuation of the CAPs. Sodium nitroprusside reduced both the CAPs and the ERG, whilst potassium ferricyanide did not affect the CAPs, and potentiated the ERG. We conclude that, independent of the vasculature, endogenous NO modulates neurotransmission (i.e. light-evoked CAPs), but probably not phototransduction (i.e. light-evoked ERG) in this intact in vitro central nervous system preparation.

Magnesium plus mexiletine inhibit energy usage and protect retinas against ischemia.

We determined whether exogenous Mg2+ and/or mexiletine (Mex), which are reported to be neuroprotective agents, reduced neuronal energy requirements and protected against ischemia, using isolated rabbit retinas. Under non-ischemic conditions, Mex (300 microM) and the combination of Mg2+ (1 mM) plus Mex (300 microM) significantly reduced glucose utilization, by 19% and 31%, respectively. The combination of Mg2+ plus Mex, but not either agent alone, significantly reduced lactate production (by 18%; p < 0.05). When added during 2 h of ischemia (simulated by the reduction of oxygen from 95% to 15% and of glucose from 6 mM to 1 mM), Mg2+ plus Mex improved the recovery of glucose utilization (p < 0.01), lactate production (p < 0.05) and neuronal function (p < 0.05) for 3 h following return to control (post-ischemia/recovery) conditions. Thus reducing energy demands by blocking functions during temporary ischemia, protects neurons from irreversible functional damage.

Nitric oxide produced during ischemia improves functional recovery in the rabbit retina.

We examined the effect of modulating endogenous nitric oxide (NO) production on the recovery of neuronal function from temporary ischemia using a preparation in which blood flow is not a factor. Inhibition of nitric oxide synthase (NOS) during ischemia with L-NA (100 mumol-1) resulted in worse functional recovery compared to D-NA (100 mumol-1)-treated in control retinas (p < 0.01). In contrast, addition of L-Arg (1000 mumol-1) during ischemia, resulted in a concentration-dependent functional improvement (p < 0.05). These results show that inhibition of constitutive NOS is detrimental, whilst the enhancement of endogenous NO production improves the recovery of neuronal function during a period of temporary ischemia in the isolated retina, (an in vitro avascular model of the CNS). Thus, independent of its effects on the vasculature, NO production during temporary ischemia protects neurons from irreversible function damage.

An in vitro rabbit retina model to study electrophysiologic and metabolic function during and following ischemia.

Most in vitro studies involving neuronal ischemia use biochemical measures and/or cell counting to assess cellular death. We describe an in vitro rabbit retina model in which we measured glucose utilization, lactate production, and light-evoked compound action potentials (CAPs) to assess metabolic and functional recovery following ischemia. Under control conditions, retinal glucose utilization and lactate production (n = 7), as well as CAPs (n = 8) remained quite constant for 6-8 h. During ischemia (glucose reduced from 6 to 1 mM and oxygen from 95 to 15%), glucose utilization and lactate production fell to 50%. CAPs fell to 50% in 3-4 min, and to 0% in 8-10 min. Recovery during 3-4 h of 'return-to-control' was dependent upon the length of ischemia. Glucose utilization recovered to 63% after 1 h (n = 4) and to 18% after 2 h of ischemia (n = 6, P < 0.001). Lactate production recovered to 77% after 1 h (n = 4) and to 54% after 2 h of ischemia (n = 6, P < 0.001). CAPs returned to 51, 15, and 0.13% of the control responses after 0.5 h (n = 7), 1 h (n = 8), and 2 h (n = 5) of ischemia, respectively (P < 0.001). This avascular, blood-brain barrier-free preparation provides an opportunity to use both metabolic and functional criteria to test protection against neuronal ischemia.

Somatostatin modulates vascular sympathetic neurotransmission in the rabbit ear artery.

Somatostatin-like immunoreactivity was localised immunohistochemically in perivascular nerves in the rabbit central ear artery. Whilst somatostatin had no direct action on this vessel, it significantly inhibited noradrenaline-induced, but not alpha, beta-methylene ATP-induced, vasoconstriction. Somatostatin also inhibited contractions elicited by electrical field stimulation showing greater effect at low (16 Hz) compared with high (64 Hz) frequencies, and inhibited the release of tritiated noradrenaline in a concentration-dependent manner. These results confirm that somatostatin is a neuroregulatory peptide, and suggest that it is modulating vascular sympathetic cotransmission of the rabbit central ear artery by acting both prejunctionally to inhibit transmitter release, and postjunctionally to reduce the action of noradrenaline.

Sensory-motor neuromodulation of sympathetic vasoconstriction in the rabbit central ear artery.

Histochemical and pharmacological studies were performed on the rabbit central ear artery. In perivascular nerves, positive immunoreactivity for calcitonin gene-related peptide and substance P was demonstrated. Calcitonin gene-related peptide-like immunoreactivity was also found to be colocalised with substance P-like immunoreactivity in a subpopulation of perivascular nerves. In vitro incubation with 6-hydroxydopamine did not alter the intensity and/or density of either the calcitonin gene-related peptide- or substance P-like immunoreactive fibres, whereas incubation with capsaicin significantly reduced both. In pharmacological studies, calcitonin gene-related peptide reduced the vasoconstrictor responses to exogenous noradrenaline and alpha, beta-methylene ATP and to electrical field stimulation in a concentration-dependent manner. In segments of the central ear artery preconstricted with noradrenaline, relaxation mediated by calcitonin gene-related peptide was endothelium-independent. These results shed new light on the innervation and nervous control of the rabbit central ear artery previously thought to be exclusively under sympathetic (adrenergic and purinergic) control. Further, the results suggest that calcitonin gene-related peptide localised in sensory nerves in the rabbit central ear artery may act as an inhibitory modulator of excitatory sympathetic vascular neurotransmission.

Neuropeptide Y potentiates purinergic as well as adrenergic responses of the rabbit ear artery.

The localisation of neuropeptide Y in the rabbit central ear artery and its pharmacological action on this preparation were investigated. Immunohistochemical studies demonstrated the presence of neuropeptide Y-like immunoreactivity in the perivascular nerves supplying the rabbit ear artery. Forty-eight hours after treatment with reserpine (5 mg/kg i.p. 48 h and 3 mg/kg i.p. 24 h prior to the experiment) catecholamine fluorescence in the rabbit central ear artery was abolished and the neuropeptide Y-like immunoreactivity was substantially reduced, suggesting that noradrenaline and neuropeptide Y were colocalised in sympathetic nerves. Contractile responses to exogenous noradrenaline (1 microM) and alpha,beta-methylene ATP (1 microM) were both significantly potentiated following incubation with neuropeptide Y (0.3 microM); the degree of potentiation being similar for both agonists. Electrical field stimulation of the rabbit central ear artery (16 and 64 Hz) produced frequency-dependent contractile responses which were abolished by tetrodotoxin (1 microM) and which were significantly potentiated in the presence of neuropeptide Y (0.3 microM). The responses to stimulation at 16 Hz were enhanced to a greater extent than the responses at 64 Hz. After blocking the noradrenergic component of the neurogenic response with prazosin (1 microM), the residual purinergic component, at both 16 and 64 Hz, was significantly enhanced in the presence of neuropeptide Y. However, following desensitisation of the P2-purinoceptor with alpha,beta-methylene ATP, neuropeptide Y had no significant effect on the residual noradrenergic component.(ABSTRACT TRUNCATED AT 250 WORDS)